In this proposal, the techniques of molecular biology will be used to analyze the tubulin genes and their expression in the unicellular organisms Polytomella and chlamydomonas. These two organisms have both flagellar and cy toplasmic microtubules in the same cell, and can therefore regulate the synthesis and assembly of two distinct microtubule types in a common cytoplasm. The research addresses two related questions: How is the expression of each of the tubulin genes regulated? and, What role, if any, does this regulation play in the generation of tubulin heterogeneity at the protein level? Copies of the tubulin genes, cloned in a lambda phage vector, will be compared by restriction endonuclease mapping and DNA sequencing techniques. Gene fragments specific for each tubulin gene will be isolated. These fragments will be used as probes to identify, for each tubulin gene, its RNA transcript(s) (by RNA blot analysis), and the tubulin protein which it encodes (by hybridization selection and in vitro translation). Studies of tubulin gene expression willinvolve changing the state of the different microtubules - e.g., by amputating the flagella or inducing disassembly of cytoplasmic microtubules - and determining how these changes alter the expression of the individual tubulin genes. The significance of this project is that it uses a simple, easily manipulated eukaryotic system to study controls on gene expression. In addition it examines the origin of microtubule diversity in eukaryotic cells by studying tubulin genes and their expression patterns.